Association between salivary immune markers and oral health conditions in pregnant women

The feasibility of using salivary immune markers to monitor physiological changes during pregnancy and their impact on oral health, particularly dental caries, is supported by recent advancements in non-invasive diagnostic methods. Saliva collection is advantageous due to its non-invasive nature, lower biosafety requirements, and ease of repeated sampling, making it ideal for longitudinal studies in pregnant women²¹.Our study provides valuable insights into the immune changes that occur during the third trimester of pregnancy, focusing on salivary immune markers and their associations with hormonal levels and microbial counts. High detection rates (> 90%) were observed for markers such as EGF, IL-1a, IL-1ra, IL-8, IL-18, MCP-1, IL-1B, and IL-6, with IL-1ra and IL-1a showing notably high mean concentrations of 2009.6 ± 2027.7 pg/mL and 1870.4 ± 2298 pg/mL, respectively. Markers like TGF-a, Eotaxin-1, and CD40L had moderate detection rates (70-87%), while IL-4, IL-5, and IL-2 had low detection rates (20-33%). Significant associations were found between oral health indicators and immune responses, with lower salivary levels of IFN-g and TNF-b in women with ≥ 4 decayed teeth and variations in immune marker levels in relation to microbial loads and fungal presence. Latent Profile Analysis identified two clusters of immune marker levels, highlighting significant differences in demographic, socioeconomic, hormonal, and microbial variables, with higher cortisol, estradiol, testosterone, T3, and T4 levels and higher counts of C. albicans and S. mutans in the low-level cluster. Logistic regression with LASSO penalty revealed significant associations between marital status, low birth weight, diabetes, hormonal levels, and microbial counts with immune marker levels, underscoring the complex interplay of factors influencing immune status during pregnancy.

The rationale behind immune markers selection

The rationale for selecting this panel of 36 immune markers is based on current literature indicating multiple immune markers associated with caries. However, in our study, we were unable to include all immune markers mentioned in literature due to constraints in sample volume and the cross-reactivity of several markers when using the same assay kit. A study by Baker et al. examined 38 immune markers among children (EGF, FGF2, CCL11(eotaxin), TGFA, CSF3, CSF2, FLT3LG, VEGFA, CX3CL1 (fractalkine), CXCL1P1, CCL7, CCL22, CXCL8, CXCL10(IP-10), CCL2, CCL3, CCL4, IFNA2, IFNG, IL-1a, IL-1b, IL-1RN, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, CD40L, TNF, and LTA) finding that ten were elevated due to dental caries¹⁷. These markers include epidermal growth factor (EGF), interleukin 10 (IL-10), colony stimulating factor 3 (CSF3), interleukin 1 receptor antagonist (IL-1RN), colony stimulating factor 2 (CSF2), CCL22, interleukin 13 (IL-13), interleukin 15 (IL-15), and interleukin 6 (IL-6), all of which have significant roles in the development and progression of dental caries¹⁷. Another cross-sectional study examined 12 immune markers (IFN-γ, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IP-10, TNF-α, VEGF-A), the concentrations of IL-1β, IL-6, IL-8, and IL-10 were markedly elevated among the caries group²². FGF expression plays a crucial role in various stages of tooth development, including the initiation of teeth and the formation of mineralized tissues. In rodents, FGFs are uniquely vital for maintaining the stem cell niche that supports the continuous growth of their incisors throughout their lives²³. Moreover, previous studies have identified elevated levels of various cytokines in caries-affected dental pulp and/or odontoblasts. These include transforming growth factor-β1 (TGFβ1), vascular endothelial growth factor (VEGF), C-C chemokine ligand 2 (CCL2/MCP1), CCL20/MIP3α, interleukin 8 (IL8/CXCL8), CXC chemokine ligand 10 (CXCL10), epithelial cell-derived neutrophil attractant 78 (ENA78), IL-1β, IL2, IL4, IL6, IL10, IL11, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)²⁴.

In adults, the selected markers include TNFα, IFN-g, GM-CSF, IL-2, IL-4, IL-6, IL-8, and IL-10, reflecting the complex interactions between the immune system and cariogenic factors in adult populations²⁵. This comprehensive panel aims to cover a broad spectrum of immune responses to provide a thorough understanding of caries-related immunological mechanisms. Furthermore, multiple studies indicated the role of IL-17 A in protection against C. albicans (an important cariogenic organism) and its regulation of antifungal immunity²⁶, IL-17 A is crucial for combating candidiasis both at mucosal and systemic levels²⁷. A single arm clinical trial on adult participants with candidiasis also revealed significant reduction in the levels of salivary cytokines Eotaxin and Fractalkine in the group receiving an antifungal intervention²⁸.

The effect of salivary hormones on the immune markers

According to Mor and Cardenas (2010), the third trimester is characterized by a renewed pro-inflammatory state, essential for initiating labor and ensuring successful delivery. This phase involves elevated levels of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6, which are crucial for processes like cervical ripening, membrane rupture, and uterine contractions²⁹. Our analysis revealed that certain salivary hormones and microbial counts are significantly associated with immune marker levels during this critical period. Specifically, higher levels of cortisol and testosterone were negatively associated with high immune markers. Cortisol functions as an immunosuppressant by downregulating key inflammatory transcription factors such as NF-kB and AP-1, while simultaneously upregulating suppressor of cytokines (SOCS). This upregulation of SOCS inhibits STAT phosphorylation, leading to a reduction in downstream pro-inflammatory gene transcription, thereby weakening the pro-inflammatory response^30,31. The observed negative association between cortisol and high immune markers is a significant finding that aligns with the well-documented immunosuppressive effects of cortisol. Cortisol, a glucocorticoid hormone, plays a crucial role in the body’s stress response and is known to suppress various components of the immune system, including cytokine production, antibody formation, and the activity of immune cells such as T-lymphocytes and macrophages³². This immunosuppressive effect can reduce the body’s ability to mount an effective immune response, which may explain why higher cortisol levels are associated with lower immune markers. Testosterone similarly exhibits immunosuppressive effects by inhibiting the production of pro-inflammatory cytokines and reducing the activity of various antimicrobial immune cells, contributing to the reduced immune marker levels³³. The results of this study are consistent with previous research, for example a study by Yang et al. (2024), which identified significant associations between salivary hormones, dental caries, and cariogenic microorganisms during pregnancy¹³. Yang et al. reported that higher levels of salivary progesterone, estradiol, testosterone, and cortisol were positively associated with increased carriage of S. mutans and higher caries risk. Our study corroborates these findings, emphasizing the role of salivary hormones in modulating oral health and immune markers.

The influence of marital status and diabetes on the immune response

Conversely, demographic factors such as being married and having diabetes were positively associated with high immune markers. The social support associated with being married may help mitigate stress and its associated immunosuppressive effects, thereby contributing to higher immune markers. Additionally, the elevated immune markers observed in married pregnant women may reflect an enhanced ability to cope with these physiological demands due to reduced stress levels³⁴. The third trimester’s pro-inflammatory state, while essential for labor, can also increase susceptibility to infections and inflammatory conditions³⁴. Thus, the supportive environment provided by a stable marital relationship might play a crucial role in balancing the immune response, potentially reducing the risk of adverse outcomes related to excessive inflammation​. Furthermore, the relationship between diabetes and altered immune marker levels during pregnancy highlights the complex interplay between metabolic and immune systems³⁵. Pregnant women with diabetes, particularly gestational diabetes mellitus (GDM) and type 1 diabetes mellitus (T1DM), demonstrate a distinctive immune profile characterized by heightened inflammation. This pro-inflammatory state is evidenced by elevated levels of biomarkers such as CRP, IL-6, and PAI-1³⁵. Hence, diabetes was positively correlated with high immune marker levels in our study cohort. Previous studies have shown that individuals with diabetes mellitus (DM) tend to have a higher incidence of both coronal and root caries, particularly when their glycemic control is inadequate³⁶. Additionally, DM is linked to changes in the immune response, which may affect salivary cytokine levels regardless of the presence of C. albicans or S. mutans.

Immune markers and low birth weight: potential predictive biomarkers

A paper by Padilla-Cáceres et al. examined the relationship between periodontal disease and adverse pregnancy outcomes, such as preterm birth and low birth weight. The study conducted a comprehensive review of systematic reviews and meta-analyses to explore this association. The findings indicated that periodontal disease in pregnant women is associated with an increased risk of preterm birth and low birth weight in newborns³⁷. This association may be due to inflammatory processes or the translocation of periodontal pathogens. This explains the relationship between poor oral health and low birth weight. An unexpected finding was evaluated in our study indicating a positive relationship between high immune markers and low birth weight, this suggests that salivary immune status in pregnant women may serve as a predictive biomarker for adverse birth outcomes, such as low birth weight. However, this potential application requires further validation through future studies to confirm its accuracy and reliability.

Immune markers in saliva as indicators of caries and microbial imbalance

Certain cytokines and chemokines have shown promise as biomarkers for caries. These immune markers are part of the body’s response to inflammation and infection, and their presence in saliva can reflect the underlying pathological processes leading to caries formation ​³⁸. The number of decayed surfaces (DS) showed a trend towards significance (p = 0.055), suggesting women with high immune markers levels have less teeth surfaces affected by caries (Table 3). Also, the number of filled surfaces (FS) shows borderline significance (p = 0.053), indicating women with high immune markers levels have less restored teeth surfaces suggesting elevated immune markers showing a protective mechanism by reducing the need for restorative treatment. Microbial analysis highlighted that higher counts of plaque C. albicans and plaque S. mutans were negatively associated with high immune markers. The significantly lower S. mutans count in dental plaque among individuals with high immune markers, despite no difference in salivary levels, suggests that host immune responses may locally modulate bacterial colonization at the site of caries initiation. This supports the ecological plaque hypothesis and highlights the potential role of immunity in maintaining oral microbial balance³⁹.

This suggests that an altered oral microbiome, potentially facilitated by a suppressed immune system due to higher cortisol and testosterone levels, can influence immune marker levels. The presence of these cariogenic microorganisms may reflect an imbalance in the oral environment due to hormonal changes and immune modulation during pregnancy.

Study limitations

The limitations of this study include the inherent constraints of a cross-sectional design, which preclude the determination of causality between salivary immune markers and the dental caries status or presence of cariogenic pathogens. Additionally, this study faced constraints in sample volume and did not account for all potential confounding variables such as dietary habits, detailed salivary characteristics, and the impact of diurnal fluctuations on immune markers. The cross-reactivity of several markers when using the same assay affected the immune markers selection process, limiting other important immune markers from being included in the analysis. The study’s participant pool was drawn exclusively from underserved communities in Upstate New York, limiting the generalizability of the findings to other populations or regions. Future research should consider longitudinal studies with more diverse populations and geographical settings to further elucidate the role of immune markers in oral health during pregnancy. Additionally, understanding the maternal influence on immunity in early life and its association with cariogenic microorganisms, as well as assessing the underlying mechanisms that link immune markers with caries risk and adverse birth outcomes, will be crucial.